The HIV-1 accessory protein Nef is essential for high-titer viral replication and AIDS progression. Nef interacts with multiple host cell signaling proteins, including the Src family kinases (SFKs) Hck, Lyn, and c-Src. Recently, we discovered drug-like compounds that target the Nef- Hck complex and block Nef-dependent HIV replication in vitro. These results show for the first time that small molecules targeting an HIV accessory protein-host cell protein kinase complex have anti-HIV activity. In light of the purpose of Program Announcement PA-06-388, the major goals of this proposal are to solve the X-ray crystal structures of Nef-SFK complexes for the first time, and to map an inhibitor binding site within these complexes. Existing Nef structures lack the N-terminal region and a large internal flexible loop critical to Nef function. Preliminary data show that this loop plays an unexpected role in SFK activation. Crystallizing a Nef-SFK complex is predicted to stabilize these flexible segments, providing the first overall view of a functional Nef conformation and identify new points of Nef-kinase contact. These structural efforts will complement ongoing work in our laboratory to understand Nef-induced Src family kinase activation as well as the mechanism of action of small molecules targeted to this complex. Our project consists of the following Specific Aims: Aim 1 (R21 phase). Determine whether Nef proteins from a wide range of HIV-1 strains are capable of SFK activation. This Aim will systematically address whether SFK binding and activation are general properties of Nef proteins representative of each of the major subgroups of HIV- 1. Recombinant Nef variants found to bind and activate SFKs in this initial screening effort will then be used in co-crystallization trials with Hck (Aim 2). Aim 2 (R21 phase). Determine the X-ray crystal structure of a Nef-SFK complex. The major objective of this Aim is to solve the crystal structure of an active Nef-Hck complex for the first time. Complex formation is anticipated to stabilize conformations of Nef and active Hck, providing the first structural insights into the active conformers of both protein partners. This structure will be crucial to our understanding of the mechanism of action of anti-retroviral compounds identified as inhibitors of the Nef-Hck complex, and will guide future rational design of new inhibitors. Aim 3 (R33 phase). Expand structural analyses to include complexes of HIV-1 Nef with other Src-family members and with newly identified HIV-1 replication inhibitors. This Aim will expand our activities to include crystal structures of the Nef-Lyn and Nef-Src complexes, which are also formed in HIV-infected cells. Comparisons of all three Nef-SFK structures will reveal key similarities that may be exploited for small molecule inhibitor design. PUBLIC HEALTH RELEVANCE: These studies are focused on the molecular structures of protein complexes that form between the HIV virulence factor Nef and host cell signaling proteins (Src-family kinases) in HIV-infected cells. Formation of these complexes is important to AIDS progression, and small molecules that interfere with their activity may provide a new approach to anti-HIV therapeutics. Solving the X-ray crystal structures of these complexes is essential to the design and development of such compounds.